Search

Gilthead seabream (Sparus aurata) is a marine finfish of economic importance in aquaculture. Despite its adaptability to varying culture conditions, gilthead seabream culture can be affected by viral, bacterial or parasitic diseases. The main route of entry of pathogens is through mucosal surfaces. Teleost external and internal surfaces are covered by mucus, mainly comprised of highly glycosylated proteins called mucins. The mucin glycans regulate pathogen growth, adhesion, virulence and inter and intra species communication. Here, we characterized the gilthead seabream mucus glycosylation, compared it to previously described species and investigated associations with microbiota. 214 glycans were identified. The majority of the glycans were found at more than one epithelial surface, but 27, 22 and 89 O-glycan structures were unique to skin, gill and intestinal sample groups, respectively. Six O-glycan core types were observed. The majority of the seabream skin and gill O-glycans were neutral with unusual poly HexNAc motifs. In contrast, seabream intestinal O-glycans were highly acidic and not of the 'poly HexNAc' type observed in skin and gill. Furthermore, gilthead seabream gill mucosa had less oligomannose and more complex N-glycans compared to skin and intestine. The concentration and diversity of bacteria was similar in skin, gill and intestine, but the bacterial species differed between epithelia and co-varied with glycan epitopes. The presence of a complex mucus glycosylation with plenty of glycan epitopes for bacterial foraging, suggest that the skin mucosal defense in seabream includes an abundant resident microbiota. This large library of structures provides a platform for further studies, for example aiming to identifying glycans to use for diagnostic purposes, to study host-microbe interactions or disease intervention therapies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Carbohydrates can both protect against infection and act as targets promoting infection. Mucins are major components of the slimy mucus layer covering the fish epithelia. Mucins can act as decoys for intimate pathogen interaction with the host afforded by binding to glycosphingolipids in the host cell membrane. We isolated and characterized glycosphingolipids from Atlantic salmon skin, gill, stomach, pyloric caeca, and intestine. We characterized the glycosphingolipids using liquid chromatography - mass spectrometry and tandem mass spectrometry and the glycan repertoire was compared with the glycan repertoire of mucins from the same epithelia. We also investigated Aeromonas salmonicida binding using chromatogram and microtiter well based binding assays. We identified 29 glycosphingolipids. All detected acid glycans were of the ganglio-series (unless shorter) and showed a high degree of polysialylation. The non-acid glycans were mostly composed of the neolacto, globo, and ganglio core structures. The glycosphingolipid repertoire differed between epithelia and the proportion of the terminal moieties of the glycosphingolipids did not reflect the terminal moieties on the mucins from the same epithelia. A. salmonicida did not bind the Atlantic salmon glycosphingolipids. Instead, we identified that A. salmonicida binding to sialic acid occurred to α2-6 Neu5Ac but not to α2-3 Neu5Ac. α2-6 Neu5Ac was present on mucins whereas mainly α2-3 Neu5Ac was found on the glycosphingolipids, explaining the difference in A. salmonicida binding ability between these host glycoconjugates. A. salmonicida´s ability to bind to Atlantic salmon mucins, but not the glycosphingolipids, is likely part of the host defence against this pathogen.

Mucins are highly glycosylated proteins that make up the mucus covering internal and external surfaces of fish. Mucin O-glycans regulate pathogen quorum sensing, growth, virulence and attachment to the host. Knowledge on this mucosal defense system can enable alternative treatments to diseases posing a threat to productivity and welfare in aquaculture. Here, we characterize the rainbow trout (Oncorhynchus mykiss) gill, skin, pyloric ceca and distal intestinal mucin O-glycosylation and compare it to known teleost O-glycomes. We identified 54 O-glycans, consisting of up to nine monosaccharide residues. Skin glycans were most acidic, shortest on average and consisted mainly of NeuAcα2-6GalNAc. Glycans from the gills were less acidic with predominantly core 1 and 2 glycans, whereas glycans from pyloric ceca and distal intestine expressed an increased number of core 5 glycans, distinctly decorated with NeuAcα2-8NeuAc- like epitopes. When compared to Atlantic salmon and Arctic charr, trends on the core distribution, average size and overall acidity remained similar, although the epitopes varied. Rainbow trout mucins from gill and intestine bound A. salmonicida and A. hydrophila more efficiently than skin mucins. This is in line with a model where skin mucins with small glycans limit bacterial adhesion to the fish surface whereas the complex intestinal mucin glycans aid in trapping and removing pathogens from the epithelial surface., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Streptococcus oralis is an oral commensal and opportunistic pathogen that can enter the bloodstream and cause bacteremia and infective endocarditis. Here, we investigated the mechanisms of S. oralis binding to oral mucins using clinical isolates, isogenic mutants and glycoconjugates. S. oralis bound to both MUC5B and MUC7, with a higher level of binding to MUC7. Mass spectrometry identified 128 glycans on MUC5B, MUC7 and the salivary agglutinin (SAG). MUC7/SAG contained a higher relative abundance of Lewis type structures, including Lewis b/y, sialyl-Lewis a/x and α2,3-linked sialic acid, compared to MUC5B. S. oralis subsp. oralis binding to MUC5B and MUC7/SAG was inhibited by Lewis b and Lacto-N-tetraose glycoconjugates. In addition, S. oralis binding to MUC7/SAG was inhibited by sialyl Lewis x. Binding was not inhibited by Lacto-N-fucopentaose, H type 2 and Lewis x conjugates. These data suggest that three distinct carbohydrate binding specificities are involved in S. oralis subsp. oralis binding to oral mucins and that the mechanisms of binding MUC5B and MUC7 differ. Efficient binding of S. oralis subsp. oralis to MUC5B and MUC7 required the gene encoding sortase A, suggesting that the adhesin(s) are LPXTG-containing surface protein(s). Further investigation demonstrated that one of these adhesins is the sialic acid binding protein AsaA ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chahal, Quintana-Hayashi, Gaytán, Benktander, Padra, King and Linden.)

Mucus, whereof the highly glycosylated mucins are a major component, protects the epithelial mucosal surfaces. The aim of this study was to characterize the rainbow trout (Oncorhynchus mykiss) gastrointestinal mucus barrier function, mucin production, glycosylation and response to lipopolysaccharide. Both gastric and intestinal mucus was thick and impenetrable to bacteria-sized beads ex vivo. The secreted mucus covering the gastric epithelium predominantly contained sialylated mucins. Plume-like structures emerging from the gastric pits were both sialylated and fucosylated, indicating heterogeneity in gastric mucus secreted by the surface mucus cells and gland secretory cells, whereas intestinal mucus appeared more homogenous. In vivo metabolic mucin labelling revealed regional differences in mucin production and basal to apical transport, while lipopolysaccharide stimulation increased the rate of mucin production and basal to apical transport in both stomach and intestine. Using mass spectrometry, 34 mucin O-glycans were identified, with ∼70% of the relative abundance being sialylated, ∼40% di-sialylated and 20-25% fucosylated. No effects of lipopolysaccharide treatment were apparent regarding O-glycan repertoires, relative abundance of components, size distribution or core structures. Thus, the mucus production and organization differ between epithelial sites but provide a barrier to bacteria in both stomach and intestine. Furthermore, mucin production and basal to apical transport was stimulated by lipopolysaccharide in all regions, suggesting a mechanism to combat infections., (Copyright © 2022. Published by Elsevier Ltd.)

Chronic obstructive pulmonary disease (COPD) is associated with colonization by bacterial pathogens and repeated airway infections, leading to exacerbations and impaired lung function. The highly glycosylated mucins in the mucus lining the airways are an important part of the host defense against pathogens. However, mucus accumulation can contribute to COPD pathology. Here, we examined whether inflammation is associated with glycosylation changes that affect interactions between airway mucins and pathogens. We isolated mucins from lower airway samples ( n  = 4-9) from long-term smokers with and without COPD and from never-smokers. The most abundant terminal glycan moiety was N -acetylneuraminic acid (Neu5Ac) among smokers with and without COPD and N -acetyl-hexoseamine among never-smokers. Moraxella catarrhalis bound to MUC5 mucins from smokers with and without COPD. M. catarrhalis binding correlated with inflammatory parameters and Neu5Ac content. M. catarrhalis binding was abolished by enzymatic removal of Neu5Ac. Furthermore, M. catarrhalis bound to α2,6 sialyl-lactose, suggesting that α2,6 sialic acid contributes to M. catarrhalis binding to mucins. Furthermore, we detected more M. catarrhalis binding to mucins from patients with pneumonia than to those from control subjects ( n  = 8-13), and this binding correlated with C-reactive protein and Neu5Ac levels. These results suggest a key role of inflammation-induced Neu5Ac in the adhesion of M. catarrhalis to airway mucins. The inflammation-induced ability of MUC5 mucins to bind M. catarrhalis is likely a host defense mechanism in the healthy lung, although it cannot be excluded that impaired mucociliary clearance limits the effectiveness of this defense in patients with COPD.

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O -glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14-30 kg/m 3 ) and high densities (HD: 50-80 kg/m 3 ) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (P app mannitol; p < 0.01). Liquid chromatography-mass spectrometry revealed 81 O -glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O -glycan structures. Overall, four of the O -glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators.

Amoebic gill disease (AGD) causes poor performance and death in salmonids. Mucins are mainly comprised by carbohydrates and are main components of the mucus covering the gill. Since glycans regulate pathogen binding and growth, glycosylation changes may affect susceptibility to primary and secondary infections. We investigated gill mucin O -glycosylation from Atlantic salmon with and without AGD using liquid chromatography-mass spectrometry. Gill mucin glycans were larger and more complex, diverse and fucosylated than skin mucins. Confocal microscopy revealed that fucosylated mucus coated sialylated mucus strands in ex vivo gill mucus. Terminal HexNAcs were more abundant among O -glycans from AGD-affected Atlantic salmon, whereas core 1 structures and structures with acidic moieties such as N -acetylneuraminic acid (NeuAc) and sulfate groups were less abundant compared to non-infected fish. The fucosylated and NeuAc-containing O -glycans were inversely proportional, with infected fish on the lower scale of NeuAc abundance and high on fucosylated structures. The fucosylated epitopes were of three types: Fuc-HexNAc-R, Gal-[Fuc-]HexNAc-R and HexNAc-[Fuc-]HexNAc-R. These blood group-like structures could be an avenue to diversify the glycan repertoire to limit infection in the exposed gills. Furthermore, care must be taken when using skin mucus as proxy for gill mucus, as gill mucins are distinctly different from skin mucins.

Diseases cause ethical concerns and economic losses in the Salmonid industry. The mucus layer comprised of highly O -glycosylated mucins is the first contact between pathogens and fish. Mucin glycans govern pathogen adhesion, growth and virulence. The Atlantic salmon O -glycome from a single location has been characterized and the interindividual variation was low. Because interindividual variation is considered a population-based defense, hindering the entire population from being wiped out by a single infection, low interindividual variation among Atlantic salmon may be a concern. Here, we analyzed the O -glycome of 25 Atlantic salmon from six cohorts grown under various conditions from Sweden, Norway and Australia (Tasmania) using mass spectrometry. This expanded the known Atlantic salmon O -glycome by 60% to 169 identified structures. The mucin O -glycosylation was relatively stable over time within a geographical region, but the size of the fish affected skin mucin glycosylation. The skin mucin glycan repertoires from Swedish and Norwegian Atlantic salmon populations were closely related compared with Tasmanian ones, regardless of size and salinity, with differences in glycan size and composition. The internal mucin glycan repertoire also clustered based on geographical origin and into pyloric cecal and distal intestinal groups, regardless of cohort and fish size. Fucosylated structures were more abundant in Tasmanian pyloric caeca and distal intestine mucins compared with Swedish ones. Overall, Tasmanian Atlantic salmon mucins have more O -glycan structures in skin but less in the gastrointestinal tract compared with Swedish fish. Low interindividual variation was confirmed within each cohort. The results can serve as a library for identifying structures of importance for host-pathogen interactions, understanding population differences of salmon mucin glycosylation in resistance to diseases and during breeding and selection of strains. The results could make it possible to predict potential vulnerabilities to diseases and suggest that inter-region breeding may increase the glycan diversity., (© 2019 Benktander et al.)

Disease outbreaks are limiting factors for an ethical and economically sustainable aquaculture industry. The first point of contact between a pathogen and a host occurs in the mucus, which covers the epithelial surfaces of the skin, gills and gastrointestinal tract. Increased knowledge on host-pathogen interactions at these primary barriers may contribute to development of disease prevention strategies. The mucus layer is built of highly glycosylated mucins, and mucin glycosylation differs between these epithelial sites. We have previously shown that A. salmonicida binds to Atlantic salmon mucins. Here we demonstrate binding of four additional bacteria, A. hydrophila, V. harveyi, M. viscosa and Y. ruckeri, to mucins from Atlantic salmon and Arctic char. No specific binding could be observed for V. salmonicida to any of the mucin groups. Mucin binding avidity was highest for A. hydrophila and A. salmonicida, followed by V. harveyi, M. viscosa and Y. ruckeri in decreasing order. Four of the pathogens showed highest binding to either gills or intestinal mucins, whereas none of the pathogens had preference for binding to skin mucins. Fluid velocity enhanced binding of intestinal mucins to A. hydrophila and A. salmonicida at 1.5 and 2 cm/s, whereas a velocity of 2 cm/s for skin mucins increased binding of A. salmonicida and decreased binding of A. hydrophila. Binding avidity, specificity and the effect of fluid velocity on binding thus differ between salmonid pathogens and with mucin origin. The results are in line with a model where the short skin mucin glycans contribute to contact with pathogens whereas pathogen binding to mucins with complex glycans aid the removal of pathogens from internal epithelial surfaces., Competing Interests: The authors have declared that no competing interests exist.

The human stomach is a harsh and fluctuating environment for bacteria with hazards such as gastric acid and flow through of gastric contents into the intestine. H. pylori gains admission to a stable niche with nutrient access from exudates when attached to the epithelial cells under the mucus layer, whereof adherence to glycolipids and other factors provides stable and intimate attachment. To reach this niche, H. pylori must overcome mucosal defence mechanisms including the continuously secreted mucus layer, which provides several layers of defence: (1) mucins in the mucus layer can bind H. pylori and transport it away from the gastric niche with the gastric emptying, (2) mucins can inhibit H. pylori growth, both via glycans that can have antibiotic like function and via an aggregation-dependent mechanism, (3) antimicrobial peptides (AMPs) have antimicrobial activity and are retained in a strategic position in the mucus layer and (4) underneath the mucus layer, the membrane-bound mucins provide a second barrier, and can function as releasable decoys. Many of these functions are dependent on H. pylori interactions with host glycan structures, and both the host glycosylation and concentration of antimicrobial peptides change with infection and inflammation, making these interactions dynamic. Here, we review our current understanding of mucin glycan and antimicrobial peptide-dependent host defence mechanisms against H. pylori infection.

Adhesion of Helicobacter pylori to the gastric mucosa is a prerequisite for the pathogenesis of H. pylori related diseases. In this study, we investigated the ganglioside composition of human stomach as the target for attachment mediated by H. pylori SabA (sialic acid binding adhesin). Acid glycosphingolipids were isolated from human stomach and separated into subfractions, which were characterized by mass spectrometry and by binding of antibodies, bacteria, and Solanum tuberosum lectin. H. pylori SabA binding gangliosides were characterized as Neu5Acα3-neolactohexaosylceramide and Neu5Acα3-neolactooctaosylceramide, while the other acid human stomach glycosphingolipids characterized (sulfatide and the gangliosides GM3, GD3, GM1, Neu5Acα3-neolactotetraosylceramide, GD1a and GD1b) were not recognized by the bacteria. Defining H. pylori binding glycosphingolipids of the human gastric mucosa will be useful to specifically target this microbe-host interaction for therapeutic intervention.

Helicobacter suis colonizes the stomach of most pigs and is the most prevalent non-Helicobacter pylori Helicobacter species found in the human stomach. In the human host, H. suis contributes to the development of chronic gastritis, peptic ulcer disease and MALT lymphoma, whereas in pigs it is associated with gastritis, decreased growth and ulcers. Here, we demonstrate that the level of H. pylori and H. suis binding to human and pig gastric mucins varies between individuals with species dependent specificity. The binding optimum of H. pylori is at neutral pH whereas that of H. suis has an acidic pH optimum, and the mucins that H. pylori bind to are different than those that H. suis bind to. Mass spectrometric analysis of mucin O-glycans from the porcine mucin showed that individual variation in binding is reflected by a difference in glycosylation; of 109 oligosaccharide structures identified, only 14 were present in all examined samples. H. suis binding to mucins correlated with glycans containing sulfate, sialic acid and terminal galactose. Among the glycolipids present in pig stomach, binding to lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer) was identified, and adhesion to Galβ3GlcNAcβ3Galβ4Glc at both acidic and neutral pH was confirmed using other glycoconjugates. Together with that H. suis bound to DNA (used as a proxy for acidic charge), we conclude that H. suis has two binding modes: one to glycans terminating with Galβ3GlcNAc, and one to negatively charged structures. Identification of the glycan structures H. suis interacts with can contribute to development of therapeutic strategies alternative to antibiotics.

Background: Pericardial tissue from various animal species is utilized for the production of the bioprosthetic heart valves (BHV) used clinically. Experimental data show that the eventual breakdown of BHV is partly due to immunological interactions with carbohydrate tissue antigens. To understand these processes, we have examined the glycolipid-based carbohydrate antigens in naïve porcine, bovine, and equine pericardia., Experimental: Total non-acid and acid glycosphingolipid fractions were isolated from porcine, bovine, and equine pericardia, and individual glycolipid compounds were characterized by thin-layer chromatography, mass spectrometry, and binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays., Results: The non-acid glycolipid fractions from all species contained glycosphingolipids based on the globo- and neolacto-series, including pentaglycosylceramides with terminal Galα3 determinants. Terminal blood group A and H (O) structures based on type 2 core chains were present in porcine pericardium, while the Forssman pentaosylceramide was found in equine pericardium. All acid glycolipid fractions contained sulfatide and several gangliosides with both N-acetyl- and N-glycolyl-neuraminic acid as terminal saccharide chain determinants., Conclusion: Several carbohydrate antigens which are potential targets for the human immune system have been identified in the animal pericardial tissues used for the production of BHV. Which of these antigens are left in the tissues after industrial BHV production processes, as well as their potential role in eventual BHV degradation, remains to be elucidated., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Gastrointestinal infections cause significant challenges and economic losses in animal husbandry. As pathogens becoming resistant to antibiotics are a growing concern worldwide, alternative strategies to treat infections in farmed animals are necessary in order to decrease the risk to human health and increase animal health and productivity. Mucosal surfaces are the most common route used by pathogens to enter the body. The mucosal surface that lines the gastrointestinal tract is covered by a continuously secreted mucus layer that protects the epithelial surface. The mucus layer is the first barrier the pathogen must overcome for successful colonization, and is mainly composed of densely glycosylated proteins called mucins. The vast array of carbohydrate structures present on the mucins provide an important setting for host-pathogen interactions. This review summarizes the current knowledge on gastrointestinal mucins and their role during infections in farmed animals. We examine the interactions between mucins and animal pathogens, with a focus on how pathogenic bacteria can modify the mucin environment in the gut, and how this in turn affects pathogen adhesion and growth. Finally, we discuss analytical challenges and complexities of the mucus-based defense, as well as its potential to control infections in farmed animals.

As a part of a systematic investigation of the species-specific expression of glycosphingolipids, acid and non-acid glycosphingolipids were isolated from three small intestines and one large intestine of the moose (Alces alces). The glycosphingolipids were characterized by binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays, and by mass spectrometry. The non-acid fractions were complex mixtures, and all had glycosphingolipids belonging to the lacto- and neolactoseries (lactotriaosylceramide, lactotetraosylceramide, neolactotetraosylceramide, Galα3-Le(x) hexaosylceramide, and lacto-neolactohexaosylceramide), globo-series (globotriaosylceramide and globotetraosylceramide), and isogloboseries (isoglobotriaosylceramide). Penta- and heptaglycosylceramides with terminal Galili determinants were also characterized. Furthermore, glycosphingolipids with terminal blood group O determinants (H triaosylceramide, H type 2 pentaosylceramide, H type 1 penta- and heptaosylceramide) were characterized in two of the moose small intestines, and in the one large intestine, while the third small intestine had glycosphingolipids with terminal blood group A determinants (A tetraosylceramide, A type 1 hexa- and octaosylceramide, A dodecaosylceramide). The acid glycosphingolipid fractions of moose small and large intestine contained sulfatide, and the gangliosides GM3, GD3, GD1a, GD1b, and also NeuGc and NeuAc variants of the Sd(a) ganglioside and the sialyl-globopenta/SSEA-4 ganglioside. In humans, the NeuAc-globopenta/SSEA-4 ganglioside is a marker of embryonic and adult stem cells, and is also expressed in several human cancers. This is the first time sialyl-globopentaosylceramide/SSEA-4 has been characterized in a fully differentiated normal tissue, and also the first time NeuGc-globopentaosylceramide has been characterized.

The x2 glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P(k)-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x2. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P1 (k) phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x2 and sialylated forms of x2, whereas x2 is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x2. Knockdown experiments with siRNA against B3GALNT1 diminished x2 levels. We conclude that x2 fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x2 joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P1 (k) or P2 (k) erythrocyte units are preferentially selected for transfusion to P(k) patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co-express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

Background: Although xenotransplantation of vascularized organs/cells has not yet reached the clinic, glutaraldehyde-treated bioprosthetic heart valves (BHV), derived from porcine or bovine tissues, are today used for clinical replacement of diseased heart valves. However, the durability of these valve cusps is limited partly due to the onset of immune responses to the grafts. The xenoantigen-determinant Galα3Gal- and corresponding anti-Gal antibodies have been postulated to in part contribute to BHV damage. However, the presence of other non-Gal carbohydrate antigen determinants as well as the immune response to these non-Gal antigens and the inflammatory response generated by their interaction with the immune system has not been studied. In this study, we have isolated and structurally characterized both non-acid and acid glycosphingolipids from naïve porcine aortic and pulmonary valve cusps., Methods: Total non-acid and acid glycosphingolipids were isolated from porcine aortic and pulmonalis valve cusps of 20 animals. Glycosphingolipid components were structurally characterized by thin-layer chromatography, liquid chromatography-mass spectrometry and binding of monoclonal antibodies and lectins., Results: The non-acid glycosphingolipids were characterized as globotetraosylceramide, H-type 2 pentaosylceramide, fucosyl-gangliotetraosylceramide, and Galα3neolactotetraosylceramide. The acid glycosphingolipid fractions had both sulfatide and gangliosides (GM3, GM2, GM1, fucosyl-GM1, GD3 and GD1a), and all gangliosides contained N-acetyl-neuraminic acid. Significantly, the N-glycolyl-neuraminic acid (NeuGc) variant, a major component in many pig organs and to which humans can develop antibodies, was not detected among the gangliosides., Conclusions: Pig valve cusps contain several complex lipid-bound carbohydrate structures that may be targets for the human immune system. Notable, the NeuGc determinant was absent in the cusp gangliosides. This work forms a platform for further characterizing the antibody reactivity of patients with porcine-derived BHV., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

One important virulence factor of enterotoxigenic Escherichia coli is their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. Here, the potential glycosphingolipid receptors of enterotoxigenic F6-fimbriated E. coli were examined by binding of purified F6 fimbriae, and F6-expressing bacteria, to glycosphingolipids on thin-layer chromatograms. When intestinal mucosal non-acid glycosphingolipids from single pigs were assayed for F6 binding capacity, a selective interaction with two glycosphingolipids was observed. The binding-active glycosphingolipids were isolated and characterized as lactotriaosylceramide (GlcNAcβ3Galβ4Glcβ1Cer) and lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer). Further binding assays using a panel of reference glycosphingolipids showed a specific interaction between the F6 fimbriae and a number of neolacto core chain (Galβ4GlcNAc) glycosphingolipids. In addition, an occasional binding of the F6 fimbriae to sulfatide, galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide was obtained. From the results we conclude that lactotriaosylceramide and lactotetraosylceramide are major porcine intestinal receptors for F6-fimbriated E. coli., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The human H-transferase (α2FucT) was introduced in Gal-negative pigs to produce pig organs not only free from Gal-antigens, but also in which the uncapped N-acetyllactosamine precursor had been transformed into non-xenogenic blood group H type 2 compounds. This work is the first descriptive analysis of glycolipids from the GalT-KO/FucT-TG pig. The aim was to investigate the cell membrane antigens in GalT-KO/FucT-TG tissues to explore its efficacy as an organ donor. Also, detailed knowledge on the correlation between the cellular glycosyltransferase configuration and the resulting carbohydrate phenotype expression is valuable from a basic glycobiological perspective., Methods: Neutral and acidic glycolipids from GalT-KO/FucT-TG small intestine were compared with glycolipids from two wildtype and two GalT-KO pig intestines. Glycolipid reactivity was tested on thin layer chromatography plates using chemical reagents, antibodies, lectins, and human serum. Structural characterization of neutral glycolipids was performed by LC-ESI/MS and proton NMR spectroscopy., Results: Characterization of the glycolipid expression in GalT-KO/FucT-TG intestine showed absence of Gal antigens and decreased/unchanged levels of the N-acetyllactosamine precursor and the blood group H type 2 expression, when compared with the wildtype. The reactivity of human serum antibodies to GalT-KO/FucT-TG derived glycolipids was similar or slightly elevated when compared with GalT-KO glycolipids. Results from LC-ESI/MS and proton NMR spectroscopy revealed no established neutral xenogenic antigens in the GalT-KO/FucT-TG pig, and could thus not explain the immunologic reactivity to human serum antibodies. The antibody binding to acidic glycolipids is most likely to be explained by the abundance of N-glycolylneuraminic acid epitopes in pig tissues. Six neutral complex biantennary glycolipids with blood group H type 1, 2, Lewis(x) and Lewis(y) determinants were found, of which three were identified in this work for the first time. One of these was a nonaglycosylceramide with blood group H type 2 and lactosyl determinants linked to a lactotetraosyl core, and the other two were decaglycosylceramides with blood group H type 1 and H type 2 determinants linked to a neolactotetraosyl core, and Lewis(x) and blood group H type 1 determinants on a lactotetraosyl core, respectively., Conclusions: Lipid-linked carbohydrate antigens in the GalT-KO/FucT-TG pig intestine showed no or minor qualitative difference when compared with GalT-KO pigs. The GalT-KO/FucT-TG pig did not appear to have an advantage over the GalT-KO pig with respect to reactivity with human antibodies from a xenotransplantation perspective., (© 2013 John Wiley & Sons A/S.)

Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 10(9) cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Le(x) pentaosylceramide, and the Le(y) hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated.

The binding of cholera toxin to the ganglioside GM1 as the initial step in the process leading to diarrhea is nowadays textbook knowledge. In contrast, the knowledge about the mechanisms for attachment of Vibrio cholerae bacterial cells to the intestinal epithelium is limited. In order to clarify this issue, a large number of glycosphingolipid mixtures were screened for binding of El Tor V. cholerae. Several specific interactions with minor complex non-acid glycosphingolipids were thereby detected. After isolation of binding-active glycosphingolipids, characterization by mass spectrometry and proton NMR, and comparative binding studies, three distinct glycosphingolipid binding patterns were defined. Firstly, V. cholerae bound to complex lacto/neolacto glycosphingolipids with the GlcNAcβ3Galβ4GlcNAc sequence as the minimal binding epitope. Secondly, glycosphingolipids with a terminal Galα3Galα3Gal moiety were recognized, and the third specificity was the binding to lactosylceramide and related compounds. V. cholerae binding to lacto/neolacto glycosphingolipids, and to the other classes of binding-active compounds, remained after deletion of the chitin binding protein GbpA. Thus, the binding of V. cholerae to chitin and to lacto/neolacto containing glycosphingolipids represents two separate binding specificities.

Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fucα2Galβ3(Fucα4)GlcNAc; Le(b)) and the H type 1 determinant (Fucα2Galβ3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Le(b) and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), and a mixture of three complex glycosphingolipids (Fucα2Galβ4GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fucα2Galβ3GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, and Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A.

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO(3)-3Galß1Cer), sulf-lactosylceramide (SO(3)-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).